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Unraveling Heparan Sulfate Proteoglycan Binding Motif for Cancer Cell Selectivity.

ABSTRACT: Membrane heparan sulfate proteoglycans (HSPG) regulate cell proliferation, migration, and differentiation and are therefore considered key players in cancer cell development processes. Here, we used the NT4 peptide to investigate how the sulfation pattern of HSPG on cells drives binding specificity. NT4 is a branched peptide that binds the glycosaminoglycan (GAG) chains of HSPG. It has already been shown to inhibit growth factor-induced migration and invasiveness of cancer cells, implying antagonist binding of HSPG. The binding affinity of NT4 with recombinant HSPG showed that NT4 bound glypican-3 and -4 and, with lower affinity, syndecan-4. NT4 binding to the cancer cell membrane was inversely correlated with sulfatase expression. NT4 binding was higher in cell lines with lower expression of SULF-1 and SULF-2, which confirms the determinant role of sulfate groups for recognition by NT4. Using 8-mer and 9-mer heparan sulfate (HS) oligosaccharides with analog disaccharide composition and different sulfation sites, a possible recognition motif was identified that includes repeated 6-O-sulfates alternating with N- and/or 2-O-sulfates. Molecular modeling provided a fully descriptive picture of binding architecture, showing that sulfate groups on opposite sides of the oligosaccharide can interact with positive residues on two peptide sequences of the branched structure, thus favoring multivalent binding and explaining the high affinity and selectivity of NT4 for highly sulfated GAGs. NT4 and possibly newly selected branched peptides will be essential probes for reconstructing and unraveling binding sites for cancer-involved ligands on GAGs and will pave the way for new cancer detection and treatment options.

SUBMITTER: Brunetti J

PROVIDER: S-EPMC6759624 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Unraveling Heparan Sulfate Proteoglycan Binding Motif for Cancer Cell Selectivity.

Brunetti Jlenia J Riolo Giulia G Depau Lorenzo L Mandarini Elisabetta E Bernini Andrea A Karousou Evgenia E Passi Alberto A Pini Alessandro A Bracci Luisa L Falciani Chiara C

Frontiers in oncology 20190918

Membrane heparan sulfate proteoglycans (HSPG) regulate cell proliferation, migration, and differentiation and are therefore considered key players in cancer cell development processes. Here, we used the NT4 peptide to investigate how the sulfation pattern of HSPG on cells drives binding specificity. NT4 is a branched peptide that binds the glycosaminoglycan (GAG) chains of HSPG. It has already been shown to inhibit growth factor-induced migration and invasiveness of cancer cells, implying antago ...[more]

PMID: 31620357

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Project description:Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes in vivo with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes in vivo, an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional in vivo. Both substitution mutations reduce AAV2's affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the in vivo function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency in vivo. The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs.

Dataset Information

Unraveling Heparan Sulfate Proteoglycan Binding Motif for Cancer Cell Selectivity.

Publications

Unraveling Heparan Sulfate Proteoglycan Binding Motif for Cancer Cell Selectivity.

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